Mitochondria are cellular organelles found in eukarytotic cells that play a central role in energy metabolism, apoptosis and ageing. Mitochondria contain a distinct mitochondrial genome, and human mitochondria contain 2 to 10 copies of their own DNA (mtDNA) of 16,569 bp, which encodes essential components of the oxidative phosphorylation machinery. This makes the mitochondrion to a certain extent independent of the nucleus, in that proteins are synthesised directly in the mitochondrion. Mitochondrial DNA resembles prokaryotic DNA in that it is a circular double stranded molecule comprising genes that do not possess introns; moreover, its genetic code differs from the “normal” universal genetic code.
The mitochondrion is highly susceptible to mutagenesis, as a result of the presence of multiple copies of mtDNA in each cell, as opposed to a single copy of nuclear DNA. Moreover, certain repair mechanisms in mitochondria are lacking, such as nucleotide excision repair mechanisms.
Point mutations, deletions or rearrangements in human mtDNA disrupt oxidative phosphorylation leading to a range of genetic diseases for which there are currently no treatments. For example, Leber hereditary optic neuropathy (LHON) is caused by a G1778A point mutation in the gene encoding the ND4 subunit of complex I in mtDNA. Neuropathy, ataxia and retinitis pigmentosa (NARP), as well as maternally-inherited Leigh syndrome (MILS) are moreover commonly resulting from a T8993G point mutation in the ATP synthase 6 gene in mtDNA. MELAS (Mitochondrial Encephalomyopathy; Lactic Acidosis; Stroke) is caused by mutations in a variety of mitochondrial genes, but usually in tRNALeu, with an A3243G mutation responsible for 80% of MELAS syndromes. MERRF (Myoclonic Epilepsy; Ragged Red Fibers) is commonly associated with a mutation in mitochondrial tRNALys; Cardiomyopathy is often associated with mutations in tRNAIle; myopathy, deafness and diabetes can also alt have mitochondrial associations.
There are still considerable uncertainties about how mtDNA is replicated, maintained and expressed. The ability to manipulate or modify particular mtDNA sequences in mitochondria within cells would facilitate investigations of normal mtDNA processes and also enable development of therapies for diseases resulting from alteration of sequences in mitochondrial DNA. However achieving this goal has proven difficult as standard gene therapy approaches such as delivering exogenous copies of DNA into mitochondria in a heritable manner remains problematic.
Although the mitochondrion comprises its own genome, it imports a large number of proteins (estimated at about 1000) which are produced on cytoplasmic ribosomes and encoded in nuclear genes. Nuclear proteins intended for import into mitochondria have signal sequences that relocate the polypeptides to the relevant organelle; these are known as mitochondrial targeting sequences (MTS).
Tanaka et al. (2002) J Biomed Sci 9:534-541 reported the delivery of a SmaI endonuclease to mitochondria by fusing it to the cytochrome c oxidase subunit IV MTS. SmaI cleaves the sequence CCC//GGG, which occurs in mtDNA as a result of a T8993G mutation, for example in NARP or MILS. The mutant mitochondrial DNA was cleaved by the restriction enzyme. Since mutant mitochondrial DNA typically coexists with wild-type mtDNA (a phenomenon known as heteroplasmy, resulting from the maternal inheritance of a plurality of mitochondria through the ovum), destruction of mutant DNA allows mitochondria comprising wild-type DNA to become dominant in the cell, and the disease condition can be reversed. However, because the specificities of naturally occurring restriction enzymes are limited in numbers, this approach is of limited application to the mutations that can be corrected.
In order to overcome such problems outside of mitochondria, fusions between the DNA-cleaving domains of restriction endonucleases and DNA-binding domains have been made. A method of converting zinc finger DNA binding domains to chimaeric restriction endonucleases has been described in Kim, et al, (1996) Proc. Natl. Acad. Sci. USA 93:1156-1160.
MTSs are known in the art and are reviewed, for example, in Pfanner & Geissler, Nature Reviews Mol Cell Biol. 2:339-49, incorporated herein by reference.
US patent application US2004/0072774 describes the use of MTS to transfer polypeptides synthesised in the cytoplasm to the mitochondrion. Examples of MTS given in US2004/0072774 include the N-terminal region of human cytochrome c oxidase subunit VIII, the N-terminal region of the P1 isoform of subunit c of human ATP synthase, and the N-terminal region of the aldehyde dehydrogenase targeting sequence.
However, US2004/0072774 is concerned with the delivery of polypeptides other than DNA-binding polypeptides. For example, US2004/0072774 suggests that mitochondrial disorders may be corrected by introducing a native mitochondrial polypeptide with the wild type sequence.
DNA binding proteins have been used to modulate gene expression in the nucleus of cells. Recombinant ZFPs have been reported to have the ability to regulate gene expression of transiently expressed reporter genes in cultured cells (see, e.g., Pomerantz et al., Science 267:93-96 (1995); Liu et al., PNAS 94:5525-5530 1997); and Beerli et al., PNAS 95:14628-14633 (1998)) and exogenous chromosomal sequences (Choo et al., Nature 372:642-645 (1994)).
More recent work shows the use of ZFP fusions to transcriptional activation and repression domains to regulate expression of endogenous chromosomal genes in their native state in cultured cells [Beerli et al. (2000) PNAS 97:1495-1500; Zhang et al. (2000) J. Biological Chemistry 275:33850-33860; Liu et al. (2001) J. Biological Chemistry 276:11323-11334] and in whole animals [Rebar et al. (2002) Nature Medicine 8:1427-1432; Dai et al. (2004) Circulation 110:2467-2475].
In particular, Beerli et al. targeted endogenous erbB-2 and erbB-3 genes using zinc fionger polypeptides produced by rational design. Using KRAB and VP64 repression and activation domains fused to the ZFPs, Beerli et al. were able to observe upregulation and downregulation of expression in endogenous erbB-2 and erbB-3 genes. Zhang et al. activated the endogenous erythropoietin gene using a ZFP linked to the VP16 transactivation domain. ZFPs were obtained by rational design and tested to determine their ability to transactivate endogenous and transfected EPO genes. ZFPs with a dissociation constant of <10 nM were found to be effective in activating transfected epo templates; a subset of these was effective in transactivating the endogenous gene. Liu et al. used ZFPs targeted to open chromatin regions in the VEGF-A gene, mapped by DNase I hypersensitivity analysis, to regulate the endogenous VEGF-A gene. Rebar et al. and Dai et al. targeted the endogenous VEGF-A gene to induce angiogenesis in a mouse model and in rabbits respectively.
ZFP fusions to the Type IIS restriction enzyme cleavage domain from FokI have been used to catalyze cleavage of chromosomal DNA at a predetermined site, promoting both targeted mutagenesis [Bibikova et al. (2002) Genetics 161:1169-1175; Lloyd et al. (2005) PNAS 102:2232-2237] and targeted homologous recombination [Porteus and Baltimore (2003) Science 300:763; Bibikova et al. (2003) Science 300:764; Urnov et al (2005) Nature 435:646-651].
Lloyd et al. used ZFPs to induce targeted mutagenesis in plant-genes. In this procedure, ZFNs were used to generate double-strand breaks at specific genomic sites. Subsequent repair by non-homologous end-joining (NHEJ) is known to be error-prone in plants and produces mutations at the break site. Constructs carrying both a ZFN gene, driven by a heat-shock promoter, and its target were introduced into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 (8%) were deletions accompanied by insertions.
Porteus and Baltimore were able to demonstrate correction of a gene defect in an exogenous integrated chromosomal GFP gene in human cells, using a ZFP-FokI fusion. Bibikova et al. showed targeted recombination, mediated by a ZFP-FokI fusion, at the yellow locus in Drosophila embryos. Umov et al. used ZFP-FokI fusion proteins to obtain targeted recombination at the endogenous IL-2Rgamma locus, in human cells, at frequencies approaching 20%. Without the ZFP fusion, targeted recombination occurs at a frequency of approximately 1-2 cells in 500,000.
None of the above constructs, however, has been used in a mitochondrion. There is therefore a need to establish whether DNA binding proteins, such as ZFPs, can be used in mitochondria; and if so, how such proteins may be targeted to the mitochondrion.